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United States Patent |
5,599,708
|
Mundy
,   et al.
|
February 4, 1997
|
Osteoclast growth regulating factors and antibodies
Abstract
The present invention relates to a novel osteoclast growth regulatory
factor, hereinafter referred to as "osteoclastpoietic factor (OPF)," which
stimulates the formation of osteoclast bone cells, a process of preparing
same, therapeutic and diagnostic uses thereof, antibodies to and
antagonists of OPF, and assays for OPF, all of which relate to the
development of therapeutics for the prevention and treatment of diseases
involving bone tissue, for example, osteoporosis, Paget's disease and
osteopetrosis.
Inventors:
|
Mundy; Gregory R. (San Antonio, TX);
Burgess; Wilson H. (Gaithersburg, MD);
Yoneda; Toshiyuka (San Antionio, TX)
|
Assignee:
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Osteosa Liquidation Trust (San Antonio, TX)
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Appl. No.:
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279675 |
Filed:
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July 25, 1994 |
Current U.S. Class: |
435/331; 424/138.1; 424/145.1; 424/155.1; 424/174.1; 424/178.1; 424/185.1; 424/198.1; 435/70.2; 435/336; 530/326; 530/388.24; 530/389.1; 530/413 |
Intern'l Class: |
C12N 015/13; A61K 039/395; C07K 014/00; C12P 021/02 |
Field of Search: |
424/138.1,145.1,155.1,158.1,174.1,178.1,185.1,198.1
435/240.3,172.2,70.2,740.27
530/326,387.7-387.9,388.24,413,391.3,389.1
|
References Cited
U.S. Patent Documents
5169837 | Dec., 1992 | Lagarde et al. | 514/21.
|
Other References
Rudinger Pepticle Harmones ed Parsons Univ. Park Press 1976 pp. 1-7.
Yoneda et al Oral Surg Oral Med Oral Pathology 68:604-611 1989.
Mundy Trends Endo. Metab. 1:307-311 1990.
Yoneda J. Clin. Oncol 9:468-477 1991.
Josse, et al J. Clin Invest G7:1472-1481 1981.
Waldmann Science 252:1657-1662 1991.
Current Protocols in Molecular Biology 1987 Wiley & Sones Chapters 10 & 11.
Yoneda et al Cancer Research 51:2438-2443 1991.
|
Primary Examiner: Budens; Robert D.
Assistant Examiner: Reeves; Julie E.
Attorney, Agent or Firm: Arnold White & Durkee
Parent Case Text
This is a continuation of U.S. patent application Ser. No. 08/059,722 filed
on 10 May 1993, now abandoned, which is a continuation of U.S. patent
application Ser. No. 07/733,420 filed on 23 Jul. 1991, now abandoned.
Claims
What is claimed as new and intended to be covered by Letters Patent of the
United States is:
1. An isolated and purified biologically active polypeptide capable of
stimulating the growth or differentiation of osteoclast cells comprising
the amino acid sequence -A-V-Q-R-Y-L-V-L-Q-G-V-S-P-A-Q-L (SEQ ID NO: 1).
2. A biologically active polypeptide according to claim 1, having a
molecular weight of from 1000 daltons to 6000 daltons as determined by gel
filtration chromatography.
3. A polypeptide according to claim 2, modified to be in detectably labeled
form.
4. An isolated and purified antibody capable of specifically binding to the
polypeptide of claim 1.
5. An antibody capable of specifically binding to the polypeptide according
to claim 1, wherein said polypeptide is isolated from the human squamous
tumor cell line MH-85, which has been deposited as ATCC number HB 10833.
6. The antibody of claim 4 which is a monoclonal antibody.
7. The antiody of claim 4 which is a polyclonal antibody.
8. A hybridoma cell line which produces the monoclonal antibody of claim 6.
9. The hybridoma cell line of claim 8 which has been deposited as ATCC
number HB 10834.
10. The polypeptide according to claim 1 purified by immunoaffinity
chromatography from the squamous tumor cell line MH-85, which has been
deposited as ATCC number HB 10833.
11. An isolated and purified antibody capable of neutralizing the
biological activity of the polypeptide of claim 1.
12. The antibody of claim 11 which is a monoclonal antibody.
13. The antibody of claim 11 which is a polyclonal antibody.
14. A biologically active polypeptide according to claim 1 having a
specific activity of at least 1 Unit per mg of protein, where Units are
defined as the number of tartrate-resistant acid phosphatase positive
multinucleated cells in the mouse marrow culture assay multiplied by
10.sup.-3.
15. A biologically active polypeptide according to claim 1 having a
specific activity of at least 4067 Units per mg of protein, where Units
are defined as the number of tartrate-resistant acid phosphatase positive
multinucleated cells in the mouse marrow culture assay multiplied by
10.sup.-3.
16. A biologically active polypeptide according to claim 1 having a
specific activity of at least 110,000 Units per mg of protein, where Units
are defined as the number of tartrate-resistant acid phosphatase positive
multinucleated cells in the mouse marrow culture assay multiplied by
10.sup.-3.
17. A hybridoma cell line which produces the monoclonal antibody of claim
12.
18. The monoclonal antibody produced by the hybridoma cell line of claim 9.
Description
FIELD OF THE INVENTION
The present invention relates to a novel osteoclast growth regulatory
factor, hereinafter referred to as "osteoclastpoietic factor (OPF)," which
stimulates the formation of osteoclast bone cells, a process of preparing
same, therapeutic and diagnostic uses thereof, antibodies to and
antagonists of OPF, and assays for OPF, all of which relate to the
development of therapeutics for the prevention and treatment of diseases
involving bone tissue, for example, osteoporosis, Paget's disease and
osteopetrosis.
Living bone tissue is continuously being replenished by the process of
resorption and deposition of calcium minerals. This process, described as,
the absorption-resorption cycle, is facilitated by means of substantially
two cell types, the osteoblasts and the osteoclasts. The osteoclast is a
multinucleated cell and is the only cell in the body known to have the
capacity to degrade (or resorb) bone. This resorption activity is
accomplished by the osteoclast forming pits (resorption lacunae) in bone
tissue, and, in fact, osteoclast activity in cell culture is measured by
their capacity to form these pits on slices of mineralized tissue such as
bone or sperm whale dentine. The osteoclast is derived from a
hematopoietic precursor which it shares with the formed elements of the
blood (Mundy & Roodman (1987) Osteoclast ontogeny and function. In Bone
and Mineral Research V: 209-280. (ed. Peck) Elsevier). The precursor for
the osteoclast is a mononuclear cell (cell with a single nucleus) which is
found in the bone marrow and which forms the mature and unique
multinucleated osteoclast after undergoing replication and differentiation
by means of cell fusion. The mature osteoclast is distinguished from other
multinucleated cells by the presence of the enzyme tartrate-resistant acid
phosphatase (TRAP) which is often used as an osteoclast cell marker.
Cells found in blood and bone respond to specific protein factors excreted
by other cells in response to various stimuli. These factors are referred
to as cytokines, many of which have been identified by their biological
characteristics and their unique amino acid sequences. Each cytokine
presents a unique spectrum of characteristics utilized to distinguish each
specific cytokine from others. Certain cytokines stimulate the growth
and/or differentiation of specific types of cells, while other cytokines
target cancerous cells for destruction. Exemplary cytokines include
granulocyte-colony-stimulating factor (CSF), granulocyte-macrophage CSF,
macrophage CSF, interleukin-1 beta, interleukin-3, interleukin-6,
interferon-gamma, tumor necrosis factor, lymphotoxin, leukemia inhibitory
factor, and transforming growth factor-alpha.
Among the pathological conditions associated with an abnormal osteoclast
development or function are conditions wherein increased bone resorption
results in the development of fragile and/or brittle bone structure, such
as osteoporosis, or increased bone absorption results in the development
of excess bone mass, such as osteopetrosis. It is believed that the
development of excess or deficient populations of osteoclasts or
osteoblasts results from a corresponding lack or excess of specific
cytokines in the blood.
Many of the known cytokines stimulate or inhibit blood cells: Several
growth regulatory cytokines such as CSF-M, transforming growth factor
alpha, interleukin-1 and tumor necrosis factor have been shown to
stimulate marrow mononuclear cell proliferation. Although cytokines such
as interleukin-1 (IL-1), tumor necrosis factor (TNF) and interleukin-6
(IL-6) may influence osteoclast formation and differentiation (Mundy
(1990) Trends Endo. Metab. 1:307-311), these factors are not specific
osteoclast growth regulatory factors.
REPORTED DEVELOPMENTS
Recently, Yoneda et al isolated a human squamous cell tumor associated with
leukocytosis and splenomegaly, hypercalcemia and increased osteoclastic
bone resorption (Yoneda et al, (1989) Oral Surgery, Oral Medicine and Oral
Pathology 68:604-611). Furthermore, when these tumors were surgically
removed, there was a dramatic decrease in osteoclastic bone resorption and
leukocyte count. Nude mice bearing the tumor also exhibited leukocytosis,
splenomegaly, hypercalcemia and increased osteoclastic bone resorption.
(Yoneda et al, 1991) J. Clin. Oncol. 9: 468-477).
The present invention relates to the isolation from the squamous tumor
cells and characterization of a polypeptide having biological activity
including regulatory activity associated with the development of
multinucleated bone cells.
SUMMARY OF THE INVENTION
The present invention relates to a biologically active polypeptide
comprising the amino acid sequence -A-V-Q-R-Y-V-L-Q-G-V-S-P-A-Q-L (SEQ ID
NO: 1) or a biologically active sequence analogue thereof. Among the
biological properties of the polypeptide of the present invention is the
capability to regulate the growth and/or differentiation of osteoclast
cells.
In another embodiment, the invention provides monoclonal and polyclonal
antibodies capable of specifically binding to the amino acid sequence
A-V-Q-R-Y-L-V-L-Q-G-V-S-P-A-Q-L (SEQ ID NO: 1) or a biologically active
sequence analogue thereof, as well as uses of these monoclonal and
polyclonal antibodies therapeutically and diagnostically. The antibodies
of the present invention are useful for affinity purifying the naturally
occurring polypeptide as well as active fragments thereof, in assays for
detecting the present polypeptide and for treating pathological conditions
resulting from over production thereof. The assays provide a method for
the clinical diagnosis and assessment of those diseases in which there is
excess production of the naturally occurring polypeptide, and for
monitoring treatment efficacy.
The invention also provides compositions, such as diagnostic and
pharmaceutical compositions, containing the polypeptide of the present
invention and methods of using these in treatment and diagnosis.
In another embodiment, the present invention provides a method for the
treatment of bone diseases characterized by abnormal osteoclast activity
such as osteopetrosis, comprising administration of the present
polypeptide to individuals in need of such treatment. Antagonists, such as
the present antibodies to the present polypeptides, are useful for
inhibiting bone resorption in a number of disease states where bone
resorption is enhanced such as, but not limited to, osteoporosis, Paget's
disease, malignant diseases which affect the skeleton such as myeloma and
breast cancer, and chronic inflammatory diseases which cause localized
bone loss such as rheumatoid arthritis and periodontal disease. Treatment
of these diseases may be accomplished by administration of antagonists
such as neutralizing antibodies to this and related polypeptides to
individuals in need of such treatment.
Other and further objects features and advantages will be apparent from the
following description of the presently preferred embodiments of the
invention, given for the purposes of disclosure when taken in conjunction
with the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
The invention will be more readily understood from a reading of the
following specification and by reference to the accompanying drawings
forming a part thereof:
FIG. 1 demonstrates the effect of MH-85CM on TRAP(+)MNC formation in mouse
bone marrow cultures.
FIGS. 2A-2D demonstrate the profile of DEAE-Cellulose anion exchange column
chromatography, of OPE (FIG. 2A), 6M-68F (FIG. 2B), 6C8F (FIG. 2C), and
M-C8F (FIG. 2D).
FIG. 3 demonstrates the profile of Sephadex-G50 gel filtration
chromatography of Peak 2 from DEAE-Cellulose column.
FIG. 4 demonstrates the profile of C8 RP HPLC column.
FIGS. 5A-5B demonstrate the effects of synthetic peptide on mouse bone
marrow cultures; effects on formation of TRAP(+) multinucleated cells
(FIG. 5A), effects on capacity to form resorption pits (FIG. 5B).
FIG. 6 demonstrates the effects of a synthetic peptide on bone resorption
in organ cultures of fetal rat long bones.
FIG. 7 demonstrates the effects of a synthetic peptide on human marrow
cultures.
FIG. 8 demonstrates the effects of different monoclonal antibodies raised
against the synthetic peptide (17-mer) on biological activity due to OPF
in MH-85 conditioned medium.
DETAILED DESCRIPTION
The present invention provides a biologically active polypeptide comprising
the amino acid sequence -A-V-Q-R-Y-L-V-L-Q-G-V-S-P-A-Q-L (SEQ ID NO: 1) or
a biologically active sequence analogue thereof. Among the biological
properties of the polypeptide of the present invention is the capability
to regulate the growth and/or differentiation of osteoclast cells.
Initially, a polypeptide capable of regulating the growth and/or
differentiation of multinucleated osteoclast cells was isolated from a
human squamous cell tumor. This polypeptide, referred to hereinafter as
osteoclastpoietic factor, or OPF, has been isolated and substantially
purified from conditioned medium from a cell line, MH-85, deposited under
ATCC Number CRL 10833, established from the human tumor described by
Yoneda et al. which was associated in the human host and in the nude mouse
host with leukocytosis, splenomegaly, hypercalcemia and increased
osteoclastic bone resorption. (Yoneda et al, 1991) J. Clin. Oncol. 9:
468-477). The present invention also provides an assay to detect the
polypeptide of the present invention. Utilizing a synthetic (17-mer)
polypeptide as a standard in an ELISA assay, OPF activity can also be
detected in the conditioned media from other animal and human cultured
tumor cells, stromal cell cultures, and media bathing murine splenic
leukocytes stimulated with the mitogen concanavalin A. (Table 1) The
limits of detection in the assay was 50 pg/ml.
TABLE 1
______________________________________
Detection of OPF from various sources.
Presence of
Cells Source OPF
______________________________________
MH-85 human squamous carcinoma
+
RWGT2 human squamous carcinoma
-
A375 human melanoma -
ST2 murine stromal cells
+
7M1 murine stromal cells
+
14M1 murine stromal cells
+
MG63 human osteosarcoma cells
-
SaOS human osteosarcoma cells
-
ROS 17/2.8 rat osteosarcoma cells
-
UMR-106 rat osteosarcoma cells
-
MC3T3 murine calvarial- bone cells
-
Con A stimulated
normal mouse spleen
+
leukocytes
______________________________________
OPF was extensively purified and isolated in a substantially homogeneous
form. OPF has a molecular weight of approximately 4000 daltons as assessed
by gel filtration chromatography. At earlier stages of purification, the
molecule having OPF activity is retained by filtration membranes which
allow molecules less than 10,000 daltons to pass. The apparent higher
molecular weight of the impure OPF might be due to aggregation of the
active moiety in these less pure preparations or to association of the OPF
molecule with another molecule.
The present invention also provides processes for purifying OPF. In one
embodiment, the present invention provides a method for purifying OPF
which comprises: collecting conditioned medium containing OPF from
cultured cells; chromatographing said medium on anion exchange medium such
as, but not limited to, DEAE-Cellulose; collecting the fractions
containing the OPF activity and subjecting the pooled fractions to gel
filtration chromatography on a Sephadex-G50 column; collecting the
fractions containing OPF activity and further subjecting the pooled
material to high pressure liquid chromatography; and recovering said OPF
in substantially pure form. Chromatographic procedures may be carded out,
preferably, in a narrow bore column containing a fine particle resin under
increased pressure to enhance the effectiveness of separation, i.e., by
high pressure liquid chromatography.
Concentration and salt removal are commonly used precursors to certain
chromatographic or separation techniques employed in the invention. Salt
removal may be performed by, for example, dialysis, gel filtration or
control pore glass (CPG) chromatography. These preparation methods and the
extent to which they are required for particular separation procedures are
well known in the art.
In another embodiment, the present invention provides a method for
purifying OPF comprising contacting a medium containing OPF mixed with
other proteins with an antibody which binds to at least one epitope of the
OPF molecule, removing the antibody-OPF complex, releasing the OPF from
the antibody and separating the OPF from the antibody. In a preferred
embodiment the antibody is bound to a solid support. The choice of solid
support and methods for binding the antibody to the solid support are well
known to those skilled in the art.
The amino acid sequence of a biologically active polypeptide which
stimulates osteoclast development has been determined. The amino acid
sequence which confers the biologic activity involved in regulating the
enhancement of multinucleated osteoclast cells comprises
-K-A-V-Q-R-Y-L-V-L-Q-G-V-S-P-A-Q-L (SEQ ID NO: 2). The present invention
also provides a synthetic biologically active analogue having the
biological activity of the naturally occurring OPF. The sequence of this
synthetic 17-mer peptide is K-A-V-Q-R-Y-L-V-L-Q-G-V-S-P-A-Q-L (SEQ ID NO:
2). The amino acid sequence of the polypeptide of the present invention is
distinct from the sequences of other proteins which have been shown to
promote osteoclast formation.
The present invention provides a biologically active polypeptide comprising
the amino acid sequence K-A-V-Q-R-Y-L-V-L-Q-G-V-S-P-A-Q-L (SEQ ID NO: 2)
in substantially homogeneous form. The term "substantially homogeneous"
when applied to the polypeptides of the present invention means that the
polypeptide is essentially free of other proteins normally associated with
OPF in its natural state. The term "substantially homogeneous" is not
meant to exclude artificial or synthetic mixtures of OPF or other
polypeptides of the present invention with other compounds.
The term "biologically active polypeptide" means naturally occurring OPF
per se, as well as biologically active analogues thereof, synthetic
produced polypeptides, natural and pharmaceutically acceptable salts and
pharmaceutically acceptable derivatives. The term "OPF" also encompasses
biologically active fragments thereof, as well as biologically active
sequence analogues thereof. Different alleles of OPF may exist in nature.
These variations may be characterized by differences in the nucleotide
sequence of the structural gene coding for proteins of identical
biological function. The term "biologically active sequence analogue"
includes analogues having single or multiple amino acid substitutions,
deletions, additions, or replacements. All such alleic variations,
modifications, and analogues resulting in derivatives of OPF which retain
one or more of the biologically active properties of native OPF are
included within the scope of this invention.
The present invention also provides a nucleotide sequence encoding the
biologically active polypeptides of the present invention. The DNA
sequence (SEQ ID NO: 3) which encodes a polypeptide (SEQ ID NO: 2) of the
present invention capable of regulating osteoclast cell differentiation
is:
##STR1##
wherein Y is selected from T or C, R is selected from A or G and N is
selected from G, A, T or C and G, A, T and C represent guanine, adenine,
thymidine, and cytosine.
The biologically active polypeptides of the present invention may also be
prepared utilizing recombinant technology. A recombinant DNA molecule
coding for any of the polypeptides of the present invention can be used to
transform a host using any of the techniques commonly known to those of
ordinary skill in the art.
A gene is a DNA sequence which encodes through its copy or messenger RNA a
sequence of amino acids characteristic of a specific peptide. The term
cDNA includes genes from which the intervening sequences have been
removed.
Methods for preparing fused, operably linked genes and expressing them in
bacteria are known and are shown, for example, in U.S. Pat. No. 4,366,246,
herein incorporated by reference. The genetic constructs and methods
described therein can be utilized for expression of the polypeptides of
the present invention in prokaryotic or eukaryotic hosts. Hosts
transformed with the polypeptides of the present invention are
particularly useful for the production of polypeptides of the present
invention
The invention extends to any host modified according to the methods
described, or modified by any other methods, commonly known to those of
ordinary skill in the art, such as, for example, by transfer of genetic
material using an expression plasmid lysogenic phage, and which yields a
prokaryote or eukaryote expressing the gene for OPF.
Especially preferred is the use of a vector containing coding sequence for
the polypeptides of the present invention for purposes of prokaryote
transformation.
The term "host" as used herein is meant to include not only prokaryotes but
also eukaryotes such as yeast as well as plant and animal cells. The term
"prokaryote" is meant to include all bacteria which can be transformed
with the DNA for the expression of the polypeptides of the present
invention. Prokaryotic hosts may include Gram negative as well as Gram
positive bacteria, such as E. coli, S. tymphimurium, Serratia marcescens,
and Bacillus subtilis. In addition to prokaryotes, eukaryotic microbes,
such as yeast cultures, may also be used. The term "eukaryote" is meant to
include all yeasts, fungi, animal and plant cells which can be transformed
with the DNA for the expression of the polypeptides of the present
invention. Eukaryotic hosts may include yeasts such as Pichia pastoris or
mammalian cells. Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among eukaryotic microorganisms, although a number of
other strains are commonly available. Yeast promoters suitable for the
expression of foreign DNA sequences in yeast include the promoters for
3-phosphoglycerate kinase or other glycolytic enzymes. Suitable expression
vectors may contain termination signals which provide for the
polyadenylation and termination of the mRNA transcript of the cloned gene.
Any vector containing a yeast-compatible promoter, origin of replication,
and appropriate termination sequence is suitable for expression of the
polypeptides of the present invention.
A cloning vehicle is a plasmid or phage DNA or other DNA sequence which is
able to replicate in a host cell which is characterized by one or a small
number of endonuclease recognition sites at which such DNA sequences may
be cut in a determinable fashion without loss of an essential biological
function of the DNA, and which contains a marker suitable for use in the
identification of transformed cells. Markers, for example, are
tetracycline resistance, neomycin resistance or ampicillin resistance. The
word "vector" is sometimes used for cloning vehicle.
An expression vehicle is a vehicle similar to a cloning vehicle but which
is capable of expressing a given structural gene in a host, normally under
control of certain control sequences. "Expression vectors" refer to
vectors which are capable of transcribing a DNA sequences contained
therein, where such sequences are linked to other regulatory sequences
capable of affecting their expression.
The expression vector typically contains an origin of replication,
promoter(s), terminator(s), a ribosome binding site, as well as specific
genes which are capable of providing phenotypic selection in transformed
cells. These expression vectors must be replicable in the host organisms
or systems either as episomes, bacteriophage, or as an integral part of
the chromosomal DNA. The transformed hosts can be fermented and cultured
according to means known in the art to achieve optimal cell growth.
Examples of promoters which can be used in the invention include, but are
not limited to: rec A, trp, lac, tac, bacteriophage lambda pR or pL, MMTV,
SV40. Examples of some of the plasmids or bacteriophage which can be used
in the invention are listed in Maniatis et al., Molecular Cloning, Cold
Spring Harbor Laboratories, 1982, and others are known to those of skill
in the art and can be easily ascertained.
Recombinant vectors and methodology disclosed herein are suitable for use
in host cells covering a wide range of prokaryotic and eukaryotic
organisms. Prokaryotic cells are preferred for the cloning of DNA
sequences and in the construction of vectors. For example, E. coli K12
strain HB101 (ATCC no. 33694), is particularly useful. Of course, other
microbial strains may be used.
Cell lines derived from multicellular organisms may also be used as hosts.
In principle, any such cell culture is workable, whether from a vertebrate
or invertebrate source. However, interest has been greatest in vertebrate
cells, and propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure in recent years. Examples of such useful hosts
are the VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI38, BHK,
COS-7, and MDCK cell lines. Expression vectors for such cells ordinarily
include an origin of replication, a promoter located in front of the gene
to be expressed, RNA splice sites (if necessary), and transcriptional
termination sequences.
For use in mammalian cells, the control functions (promoters and enhancers)
on the expression vectors are often provided by viral material. For
example, commonly used promoters are derived from polyoma, Adenovirus 2,
and most frequently, Simian Virus 40 (SV40). Eukaryotic promoters, such as
the promoter of the murine metallothionein gene [Paulakis and Hamer, Proc.
Natl. Acad. Sci. 80:397-401 (1983)], may also be used. Further, it is also
possible, and often desirable, to utilize the promoter or control
sequences which are naturally associated with desired gene sequence,
provided such control sequences are compatible with the host system. To
increase the rate of transcription, eukaryotic enhancer sequences can be
obtained from a variety of animal cells or oncogenic retroviruses such as
the mouse sarcoma virus.
An origin of replication may be provided either by construction of the
vector to include an exogenous origin, such as that provided by SV40 or
other viral sources, or may be provided by the host cell chromosomal
replication mechanism. If the vector is integrated into the host cell
chromosome, the latter is often sufficient.
Host cells can prepare the polypeptides of the present invention which can
be of a variety of chemical compositions. The polypeptide may be produced
having methionine as its first amino acid. This methionine is present by
virtue of the ATG start codon naturally existing at the origin of the
structural gene or by being engineered before a segment of the structural
gene. The protein may also be intracellularly or extracellularly cleaved,
giving rise to the amino acid which is found naturally at the amino
terminus of the polypeptide. The polypeptide may be produced together with
either its own or a heterologous signal peptide, the signal polypeptide
being specifically cleavable in an intra- or extracellular environment.
Finally, the polypeptides of the present invention may be produced by
direct expression in mature form without the necessity of cleaving away
any extraneous polypeptide.
Recombinant host cells refer to cells which have been transformed with
vectors constructed using recombinant DNA techniques. As defined herein,
the polypeptides of the present invention is produced as a consequence of
this transformation. The polypeptides of the present invention or
fragments thereof produced by such cells are referred to as "recombinant
polypeptides of the present invention."
Several groups of workers have isolated mixtures of messenger RNA (mRNA)
from eukaryotic cells and employed a series of enzymatic reactions to
synthesize double-stranded DNA copies which are complementary to this mRNA
mixture. In the first reaction, mRNA is transcribed into a single-stranded
complementary DNA (cDNA) by an RNA-directed DNA polymerase, also called
reverse transcriptase. Reverse transcriptase synthesizes DNA in the 5'-3'
direction, utilizes deoxyribonucleoside 5'-triphosphates as precursors,
and requires both a template and a primer strand, the latter of which must
have a free 3'-hydroxyl terminus. Reverse transcriptase products, whether
partial or complete copies of the mRNA template, often possess short,
partially double-stranded hairpins ("loops") at their 3' termini. In the
second reaction, these "hairpin loops" can be exploited as primers for DNA
polymerases. Preformed DNA is required both as a template and as a primer
in the action of DNA polymerase. The DNA polymerase requires the presence
of a DNA strand having a free 3'-hydroxyl group, to which new nucleotides
are added to extend the chain in the 5'-3' direction. The products of such
sequential reverse transcriptase and DNA polymerase reactions still
possess a loop at one end. The apex of the loop or "fold-point" of the
double-stranded DNA, which has thus been created, is substantially a
single-strand segment. In the third reaction, this single-strand segment
is cleaved with the single-strand specific nuclease S1 to generate a
"blunt-end" duplex DNA segment. This general method is applicable to any
mRNA mixture, and is described by Buell, et al., J. Biol. Chem., 253:2483
(1978).
The resulting double-stranded cDNA mixture (ds-cDNA) is inserted into
cloning vehicles by any one of many known techniques, depending at least
in part on the particular vehicle used.
In general, methods can be found in Maniatis, et al., supra, and Methods In
Enzymology, Volumes 65 and 68 (1980); and 100 and 101 (1983). In general,
the vector is linearized by at least one restriction endonuclease, which
will produce at least two blunt or cohesive ends. The ds-cDNA is ligated
with or joined into the vector insertion site.
Once the DNA segments are inserted, the cloning vehicle is used to
transform a suitable host. These cloning vehicles usually impart an
antibiotic resistance trait on the host. Such hosts are generally
prokaryotic cells.
If prokaryotic cells or other cells which contain substantial cell wall
material are employed, the most common method for transformation with the
expression vector is calcium chloride pretreatment as described by Cohen,
R. N., et al., Proc. Nat'l. Acad. Sci. USA, 69:2110 (1972). If cells
without cell wall barriers are used as host cells, transfection is carried
out by the calcium phosphate precipitation method described by Graham and
Van der Eb, Virology, 52:456 (1973). Other methods for introducing DNA
into cells such as electroporation, nuclear injection, viral infection or
protoplast fusion may be successfully used. At this point, only a few of
the transformed or transfected hosts contain the desired cDNA. The sum of
all transformed or transfected hosts constitutes a gene "library". The
overall ds-cDNA library created by this method provides a representative
sample of the coding information present in the mRNA mixture used as the
starting material.
Clones containing part or the entire cDNA for the the polypeptides of the
present invention are identified with specific oligonucleotide probes
deduced from a partial amino acid sequence determination of the
polypeptides of the present invention. This method of identification
requires that the non-degenerate oligonucleotide probe be designed such
that it specifically hybridizes to the cDNA corresponding to the present
invention. Clones containing cDNA sequences encoding the polypeptides of
the present invention are isolated as follows.
Individual transformed or transfected cells are grown as colonies on a
nitrocellulose filter paper. These colonies are lysed; the DNA released is
bound tightly to the filter paper by heating. The filter paper is then
incubated with a labeled oligonucleotide probe which is complementary to
the structural gene of interest. The probe hybridizes with the cDNA for
which it is complementary, and is identified by autoradiography. The
corresponding clones are characterized in order to identify one or a
combination of clones which contain all of the structural information for
the desired protein.
Clones containing additional sequence may also be identified using as probe
the cDNA insert isolated during the initial screening of the cDNA library.
Nucleotide sequencing techniques are used to determine the sequence of
amino acids encoded by the cDNA fragments.
The nucleic acid sequence coding for the protein of interest is isolated
and reinserted into an expression vector. The expression vector brings the
cloned gene under the regulatory control of specific prokaryotic or
eukaryotic control elements which allow the efficient expression
(transcription and translation) of the ds-cDNA. Thus, this general
technique is only applicable to those proteins for which at least a
portion of their amino acid or DNA sequence is known for which an
oligonucleotide probe is available. See, generally, Maniatis, et al.,
supra.
More recently, methods have been developed to identify specific clones by
probing bacterial colonies or phage plaques with antibodies specific for
the encoded protein of interest. This method can only be used with
"expression vector" cloning vehicles since elaboration of the protein
product is required. The structural gene is inserted into the vector
adjacent to regulatory gene sequences that control expression of of the
protein. The cells are lysed, either by chemical methods or by a function
supplied by the host cell or vector, and the protein is detected by a
specific antibody and a detection system such as enzyme immunoassay. An
example of this is the lambda gt.sub.11 system described by Young and
Davis, Proc. Nat'l. Acad. Sci. USA, 80:1194-1198 (1983) and Young and
Davis, Science, 222:778 (1983).
By providing the DNA sequences, and recombinant DNA molecules, the present
invention also provides probes and methods to identify cells containing or
lacking these sequences, and means to administer these sequences to cells.
This will enable the establishment of systems in which the recombinant
protein is produced after transfection of an expression vector into
appropriate host cells. Additionally, the present invention provides a
means to inhibit the expression of the novel sequences by providing an
antisense RNA sequence which, when administered to a cell, or when the DNA
encoding said antisense RNA is administered to a cell, said DNA sequence
will produce an antisense RNA which can bind to and therefore block the
expression of the RNA encoding the novel polypeptides of the present
invention. It will also be apparent to one of skill in the art from this
disclosure that antibodies against any of the proteins of the present
invention can be utilized to block the binding of ligands to the
polypeptides and to target drugs or other agents (such as labels) to the
cells expressing these polypeptides.
Monoclonal antibodies of the present invention may be prepared using the
method of Mishell, B. B., et al., Selected Methods In Cellular Immunology,
(W. H. Freeman, ed.) San Francisco (1980). The 17-mer synthetic peptide
was used as the antigen for the production of these antibodies. Briefly, a
synthetic 17-mer OPF peptide was used to immunize spleen cells of Balb/C
mice. The immunized spleen cells were fused with FO myeloma cells. Fused
cells containing spleen and myeloma cell characteristics were isolated by
growth in HAT medium, a medium which kills both parental cells, but allows
the fused products to survive and grow.
The anti-OPF antibodies are useful in the treatment of disease states
caused by increased levels in the individual of OPF. These treatments
include administration of anti-OPF monoclonal antibodies to individuals
suffering from osteoporosis, malignant diseases which affect the skeleton
such as myeloma and breast cancer, and chronic inflammatory diseases which
cause localized bone loss such as rheumatoid arthritis and periodontal
disease. These antibodies are also useful in assays for detecting or
quantitating levels of OPF. These assays provide a clinical diagnosis and
assessment of those diseases in which excess production of these factors
occurs, and a method for monitoring treatment efficacy. Synthetic
antagonists to OPF have the same beneficial therapeutic effect as
neutralizing antibodies in those diseases with this overproduction of OPF.
Neutralizing antibodies will inhibit the activity of the excessively
produced OPF in these individuals.
The term "individual" is meant to include any animal, preferably a mammal,
and most preferably a rodent, cat, dog, cow or human.
The techniques for detectably labelling the homogeneous OPF and the
monoclonal antibodies thereto of the present invention with a radiolabel,
an enzyme label, or a fluorescent label are well known to those of skill
in the art. Reference can be made to Chard, An Introduction To
Radioimmunoassay And Related Techniques, North-Holland Publishing Co.,
Amsterdam-NY-Oxford (1978), The Enzyme-Linked Immunoadsorbent Assay
(ELISA) by Voller, A., et al., Dynatech Europe Borough House, Rue du Pre,
Guernsey, Great Britain, and Radioiodination Techniques, Review 18,
Amersham Corporation, by A. E. Bolton, all incorporated herein by
reference. Preferably, the purified OPF is labelled with .sup.125 I using
the Bolton/Hunter reagent which involves succinylation of the free
N-terminals and lysine. DNA probes may also be labeled with a detectable
label. Commonly used detectable labels are radioactive labels including,
but not limited to, .sup.32 P, .sup.14 C, .sup.125 I, .sup.3 H and .sup.35
S. Biotin labeled nucleotides can be incorporated into DNA or RNA by nick
translation, enzymatic, or chemical means. The biotinylated probes are
detected after hybridization using avidin/streptavidin, fluorescent,
enzymatic or colloidal gold conjugates. Nucleic acids may also be labeled
with other fluorescent compounds, with immunodetectable fluorescent
derivatives or with biotin analogues. Nucleic acids may also be labeled by
means of attaching a protein. Nucleic acids cross-linked to radioactive or
fluorescent histone HI, enzymes (alkaline phosphatase and peroxidases), or
single-stranded binding (ssB) protein may also be used.
OPF to which there has been added a detectable label, such as a radioactive
label such as 125 I, a fluorescent label such as fluorescein or rhodamine,
biotin for non-covalent linking, or an enzymatic label such as alkaline
phosphatase, a linking group, such as SASD (sulfosuccinimidyl
2-(p-azidosalicylamido) ethyl-1,3'-dithiopropionate a protecting or
blocking group useful for specific reactive side groups during further
chemical reactions or diagnostic assays, such as the omega-amino group of
lysine. This can achieved using acylation and can be de-blocked using HCI
and dioxane or neat trifluoroacetic acid plus thioanisole. Tyrosine can be
modified by converting to amino tyrosine with tetranitromethane and sodium
hydrosulfide. Other derivatives may also be prepared from the functional
groups which occur at side chains on the residues of the N- or C-terminal
groups, by means known in the art.
Administration of the compounds useful in the method of present invention
may be by parenteral, intravenous, intramuscular, subcutaneous, rectal or
any other suitable means. The dosage administered may be dependent upon
the age, weight, kind of concurrent treatment, if any, and nature of the
pathological state being treated. The effective compound useful in the
method of the present invention may be employed in such forms as capsules,
liquid solutions, suspensions or elixirs for oral administration, or
sterile liquid forms such as solutions or suspensions. Any inert carrier
is preferably used, such as saline, or phosphate-buffered saline, or any
such carrier in which the compounds used in the method of the present
invention have suitable solubility properties for use in the method of the
present invention.
As used herein the term "salts" refers to both salts of carboxy groups of
the polypeptide or protein chain and to acid addition salts of amino
groups of the polypeptide chain. Salts of the carboxy group may be formed
with either inorganic or organic bases by means known in the art per se.
Inorganic salts include, for example, sodium, calcium, ammonium, ferric or
zinc salts, and the like. Salts with organic bases include those formed,
for example, with amines such as triethanolamine, arginine, lysine,
piperidine, caffeine, procaine and the like. Acid addition salts include,
for example, salts with mineral acids such as, for example, hydrochloric
acid or sulfuric acid, and salts with organic acids such as, for example,
acetic acid or oxalic acid.
Both the salts and the derivatives encompassed by the invention include
those which are therapeutically or diagnostically acceptable, i.e., those
which do not destroy the biologic, immunogenic, or binding activity of OPF
depending on the functional activity desired to be utilized.
The term "specific activity" refers to the activity of OPF in OPF assays
described in this application and known in the art related to the amount
of protein by weight in the sample. As specified in the current
disclosure, the activity of OPF is measured according to the assay
procedures set forth hereinbelow in Examples 1, 2, and 5. Specific
activity is calculated as shown in Example 3.
Having now generally described the invention, a more complete understanding
can be obtained by reference to the following specific examples. These
examples are provided for the purpose of illustration only and are not
intended to be limiting unless otherwise specified.
EXAMPLE 1
Assay of OPF in Conditioned Medium from MH-85 Cells
The origin of the MH-85 tumor cells has been reported (Yoneda et al, (1991)
J. Clin. Invest. 87:977-985; J. Clin. Oncol. 9:468-477). MH-85 cells were
established in culture after isolation from a human squamous cancer of the
maxilla. Six days after these cells are plated with an initial inoculation
of 5.times.10.sup.5 cells/2 ml well, 26.times.10.sup.5 cells are present.
The doubling time is approximately 28 hrs.
A modification of mouse bone marrow cultures developed by Takahashi et al
was used to assess OPF activity in MH-85 CM and fractionated
samples(Takahashi et al (1988) Endocrinology 122:1373-1382). Four to
6-week old male ICR Swiss mice (Harlan Industries, Houston, Tex.) were
killed by cervical dislocation and immersed briefly in 80% ethanol. The
femora and tibiae were removed aseptically and dissected free of adherent
tissues. The bone ends were removed with a scalpel and the marrow cavity
was flushed through with .alpha.MEM using 27G needles. Marrow cells from 3
to 5 mice were collected, washed twice with .alpha.MEM and incubated for 2
h on tissue culture plates. Nonadherent cells were resuspended in
.alpha.MEM supplemented with 10% FCS (Hyclone, Logan, Utah) and no
antibiotics at a final density of 4.times.10.sup.6 cells per ml. Half a ml
of cell suspension/well was inoculated onto 24 well plates (Corning,
Corning, N.Y.) and 1,25-dihydroxyvitamin D.sub.3 (1,25D.sub.3) (Hoffman-La
Roche, Nutley, N.J.) was added to each well at a final concentration of
10.sup.-8 M. Appropriate dilutions of MH-85 CM or column fractions (5-25%)
were added to the wells. Cultures were maintained at 37.degree. C. in a
humidified atmosphere of 5% CO.sub.2 in air. The cells were fed every 2
days with 0.3 ml of fresh .alpha.MEM with 10% FCS and 10.sup.-8 M
1,25D.sub.3. After 6 days, the cultures were washed in PBS, fixed in 60%
acetone in citrate buffer pH 5.4 for 60 seconds, air dried and stained for
tartrate resistant acid phosphatase (TRAP) using a commercially available
kit (Sigma Chemical Co., St. Louis, Mo.). Multinuclearity and TRAP are
characteristic properties of osteoclasts. Thus, the osteoclastic nature of
the cells is confirmed by these parameters. Stained cultures were examined
under light microscopy at .times.200 magnification. TRAP positive (red
staining) multinucleated (3 or more nuclei) cells (TRAP(+)MNC) were easily
distinguishable from other cells present. All TRAP(+)MNC in each well were
counted by manually scanning across the entire well in a systematic
fashion.
MH-85 cells were grown in .alpha.-minimal essential medium (.alpha.MEM)
supplemented with 5% fetal calf serum (FCS, Hazleton, Lenexa, Kans.) and
1% penicillin-streptomycin solution (GIBCO, Grand Island, N.Y.).
Serum-free culture supernatants of MH-85 (MH-85CM) were harvested from 48
h incubation of subconfluent MH-85 cultures. As a control, conditioned
media (CM) was harvested from another human squamous cancer of the maxilla
(OKK) which was not associated with leukocytosis, hypercalcemia and
cachexia. MH-85 conditioned media contained an activity which stimulates
the formation of multinucleated cells from mouse mononuclear bone marrow
cells, which comprise mononuclear precursors of osteoclasts. MH-85 CM
caused a marked increase in the formation of multinucleated osteoclasts in
these cultures, as assessed by the presence of tartrate-resistant acid
phosphatase (TRAP) and their capacity to form pits (resorption lacunae) on
a bone substitute (sperm whale dentine). FIG. 1 demonstrates the effect of
MH-85 CM on tartrate-resistant acid phosphatase in multinucleated cells
TRAP(+)MNC formation in mouse bone marrow cultures. Serum-free MH-85 CM
was harvested from 48 h incubation of sub-confluent cultures, centrifuged
and assayed in mouse bone marrow cultures (2.times.10.sup.6 cells/0.5
ml/well, 24-well plates) in the presence or absence of 10.sup.-8 M,
1,25-dihydroxyvitamin D.sub.3 (1,25D.sub.3). 1,25D.sub.3 is added as a
differentiation factor for this assay. After 6 days, cells were stained
for TRAP activity and numbers of TRAP(+)MNC were counted. Each value
represents the mean.+-.S.E. of 4 wells. There was a statistically
significantly increase in TRAP-positive multinucleated cells (cells with
osteoclast characteristics) in the cultures in response to MH-85 CM, both
in the presence and absence of 1,25D.sub.3 (p<0.01 Student's T-test). One
asterisk indicates that a value is significantly different from the
control. Two asterisks indicates that a value is significantly different
from cultures treated with 1,25D.sub.3 or MH-85CM alone.
This stimulatory effect was dose-dependent and maximal at 20% of CM
concentration. This effect was enhanced in the presence of 10.sup.-8 M
1,25D.sub.3. CM from another human tumor (OKK) which was not associated
with increased osteoclastic bone resorption, hypercalcemia and
leukocytosis had no effect.
EXAMPLE 2
Assay for Bone-Resorbing Activity of TRAP(+)MNC (Multinucleated Cells with
Osteoclast Characteristics)
Quantitation of the effects of TRAP(+)MNC on calcified matrices (sperm
whale dentine) was performed using minor modifications of the
disaggregated osteoclast resorption assay, as described by Boyde et al
(1984) Br Dent J 156:216. Mouse bone marrow cells, prepared as described
above, were cultured in .alpha.-MEM supplemented with 10% FCS on slices of
sperm whale dentine for 8 days in the presence of 10.sup.-8 M 1,25D.sub.3
and MH-85 CM or partially-purified factors. Slices of sperm whale dentine
(0.25.times.10.times.8 mm) were prepared using a Buehler low speed diamond
saw (Buehler, Lake Bluff, Ill.), followed by sonication (15 min) in
several changes of distilled water. Slices were smoothed between 2 glass
plates and sterilized under UV light for 4 days. Before all experiments,
slices were incubated in .alpha.MEM supplemented with 10% FCS and 1%
penicillin-streptomycin solution (GIBCO) for at least 48 h. Cultures were
performed in 24-well microtiter plates in humidified air (10% CO.sub.2) at
37.degree. C. (1 slice/well). A single experiment used 16-20 slices, with
a minimum of 3-4 slices/treatment. After 8-days of culture, some of the
slices with cells stained for TRAP and TRAP(+)MNC were examined under
light microscopy. For examination of resorption lacunae (pits), the slices
were sonicated in 0.1M NaOH and stained with 0.1% (IV/V) toluidine blue.
Lacunae were examined using light microscopy, and the plan area of matrix
resorbed was quantitated using a computer-assisted morphometric program on
a Quantex image and process analysis system (Quantex Instruments,
Sunnybrook, Calif.). Data were analyzed by pattern analysis of variance
(Oreffo et al, (1990) Endocrinology 126:3069-3075).
EXAMPLE 3
Purification of OPF
One hundred liters of serum-free MH-85 CM was collected, concentrated by
ultrafiltration using a Pellicon cassette (Millipore, Bedford, Mass.) with
a filter of nominal molecular weight cut-off of 10,000 to 2.5 liter and
precipitated with 80% (W/V) ammonium sulfate. The precipitate was
dissolved in 180 ml distilled water and dialyzed against distilled water
[16 liters for about 24 hours] and then against 10 mM sodium phosphate
buffer (pH 8.3)[3 changes of 16 liters for about 24 hours per change].
The concentrated and dialyzed MH-85 CM was fractionated by anion exchange
chromatography on a 4.8.times.30 cm DEAE-Cellulose DE-52 (Whatman,
Hillsboro, Oreg.) column equilibrated with 10 mM phosphate buffer (pH 8.3)
at a flow rate of 90 ml/h using a step-wise gradient of sodium phosphate
buffer. Twenty ml fractions were collected and aliquots (1 ml) were tested
for TRAP(+)MNC formation as described in Example 1. There were 2 peaks of
activity which stimulated TRAP(+)MNC formation in the mouse bone marrow
culture assay. This activity was shown to be effected by OPF. The 2nd
peak, which eluted at a different position from other known colony
stimulating factors, such as GM-CSF, G-CSF and M-CSF, eluted between 0.04
and 0.06M phosphate (FIG. 2). FIG. 2 demonstrates the profile of the
DEAE-Cellulose anion exchange column chromatography. Aliquots (2 ml) were
taken from each fraction (20 ml/fraction), dialyzed, lyophilized,
reconstituted in culture medium and assessed for their OPF activity in
mouse marrow culture assay in the presence of 10.sup.-8 M 1,25D.sub.3 and
CSF activity in human marrow culture colony forming assay. The total
activity of the polypeptide was determined by counting the number of
TRAP(+) multinucleated cells at the end of the culture period and
multiplying the total number of positive cells by 10.sup.4 (since 0.01% of
100 I total starting sample was assayed). Specific activity was calculated
as total activity divided by amount of protein in mg multiplied by
10.sup.-3. Table 2 demonstrates that at least a 4519 fold purification was
obtained following DEAE chromatography.
TABLE 2
__________________________________________________________________________
Purification of OPF
Total
Total
Protein
Activity
Specific
Fold- Recovery
Purification Step
(mg) Cells .times. 10.sup.-4
Activity
Purification
%
__________________________________________________________________________
Serum-free MH-
1820 170 0.9 1 100
85CM
Concentration
1526 161 1.1 1 95
10kD cut off
membrane
80% (NH.sub.4).sub.2 SO.sub.4
1247 139 1.1 1 82
Precipitation
DE-52 Column
0.3 122 4067 4519 72
Gel Filtration
0.01 34 34,000
37800
20
Sephadex 6-50
RP HPLC 0.003
33 110,000
122,200
19
__________________________________________________________________________
The pooled fractions of peak 2 were further purified using gel filtration
chromatography. The active fractions obtained from DEAE-Cellulose
chromatography were pooled, dialyzed overnight against 50 mM ammonium
bicarbonate (pH 7.2) and lyophilized. (Approximately 200 mls of pooled
fractions were dialyzed against 4 changes of 16 liters of dialysis
buffer). The lyophilized material was dissolved in approximately 0.5 ml of
50 mM ammonium bicarbonate and fractionated by size exclusion
chromatography on a 2.5.times.60 cm Sephadex G-50 column. (Pharmacia,
Piscataway, N.J.) column equilibrated with 50 mM ammonium bicarbonate at a
flow rate of 30 ml/h. A broad peak of activity which stimulated the
formation of TRAP(+)MNC was eluted at positions of molecular weight of
about 4 kD on a Sephadex G-50 column (FIG. 3). FIG. 3 demonstrates the
profile of Sephadex-G50 gel filtration chromatography of Peak 2 from
DEAE-Cellulose column. Aliquots (1 ml) were taken from each fraction (10
ml/fraction), lyophilized, reconstituted in .alpha.MEM supplemented with
10% FCS and assayed for their OPF activity in mouse marrow culture assay
in the presence of 10.sup.-8 M 1,25D.sub.3.
Standard molecular weight marker proteins used were blue dextran (void
volume), ribonuclease (14K), insulin (6K), a peptide synthesized in our
laboratory of 1 k, and phenol red (0.3K).
When mouse bone marrow cells were cultured on sperm whale dentine in the
presence of 10.sup.-8 M 1,25D.sub.3 and with or without the active peak
from Sephadex-G50 chromatography, characteristic resorption pits were
found. In the presence of this peak, much greater numbers of TRAP(+)MNC
and increased area of resorption lacunae (pits) were seen. These findings
demonstrated that the active peak contained a biological activity which
increases the formation of mouse multinucleated cells with osteoclast
characteristics in long-term marrow cultures.
This first part of this active peak (peak A from FIG. 3) was further
chromatographed on a C8 reverse phase HPLC analytical column
(2.1.times.150 mm) with a gradient of 0.1% TFA and 0.1% TFA/70%
acetonitrile at a flow rate of 0.15 ml/min. The absorbance was monitored
at 214 nm. FIG. 4 demonstrates the elution profile of C8 RP HPLC column.
To measure the biological activity in HPLC fractions, an aliquot of 5% of
each fraction was lyophilized in the presence of carrier BSA and
reconstituted in cell culture media. Activity was then assayed on marrow
mononuclear cell cultures. The biologically active fraction containing
osteoclastpoietic activity eluted from this column at 38% acetonitrile (45
min).
The final product had a specific activity of 110,000 demonstrating a total
purification greater than 120,000 fold.
EXAMPLE 4
Synthesis of Biologically Active OPF polypeptide (17-mer)
The fraction from the HPLC purification of Example 3 containing the
biological activity was digested with Lys-C according to the method of
(Matsudaira P: Limited N-terminal sequence analysis. In Guide to Protein
Purification (ed. Deutscher MP), Methods in Enzymology, Vol. 182, Academic
Press, Inc., San Diego, pp. 602-612, 1990.). Resulting peptide fragments
were isolated by reverse phase microbore HPLC under the same conditions as
described in Example 3. The amino acid sequence of one of the fragments
was established using a pulsed-liquid gas-phase microsequencer (Applied
Biosystems gas-phase microsequencer).
A novel peptide of 18 amino acids was obtained following enzyme digestion.
The amino acid sequence was - K-A-V-Q-R-Y-L-V-L-Q-G-V-S-P-A-Q-L, (SEQ ID
NO: 2). The first amino acid (K) is assumed, based on knowledge of the
enzymatic properties of Lys-C.
A 17-mer synthetic peptide was prepared having the same amino acid
sequence, using an Applied Biosystems 480A peptide synthesizer. When the
activity of the synthetic peptide was tested in mouse bone marrow
cultures, it produced identical effects to those seen with MH-85
conditioned media or partially purified OPF fractions. FIG. 5 demonstrates
that 2 ng/ml of the synthetic peptide increased the formation of
multinucleated cells which were TRAP positive (panel 5a), and formed
resorption lacunae (panel 5b). This peptide was active in concentrations
of 0.01-2 ng/ml.
EXAMPLE 5
Effects of the Synthetic Peptide
The synthetic peptide was tested in the same assays as the original
conditioned media. It was initially tested on the formation of
multinucleated cells with osteoclast characteristics (staining with
tartrate-resistant acid phosphatase) in murine marrow cultures. At
concentrations of 2 ng/ml, the synthetic peptide caused a profound
increase in the formation of TRAP-positive multinucleated cells. In
addition, the effects of the synthetic peptide were tested for the
capacity to stimulate murine marrow cultures to form multinucleated cells
which have the capacity to form resorption pits on calcified matrix (sperm
whale dentine). FIG. 6 demonstrates the effect of the synthetic 17-mer OPF
peptide (10 ng/ml) on bone resorption as assessed by .sup.45 Ca release
from fetal rat long bones in organ culture. There was a similar increase
in the number and area of resorption pits as that seen when murine marrow
cultures were cultured with the conditioned media from MH-85 cultures. The
peptide was also tested in other bone resorption assays, including the
capacity to stimulate the formation of cells with osteoclast
characteristics from human bone marrow cultures and its capacity to
stimulate bone resorption in organ cultures of fetal rat long bones. It
was effective in both of these assays in concentrations between 2-10
ng/ml. FIG. 7 demonstrates the effects of the 17-mer synthetic OPF peptide
on human osteoclast cell formation at doses ranging from about 0.01 to
about 10 ng/ml. Maximal activity was observed at about 2 ng/ml.
EXAMPLE 6
Purification of OPF Polyclonal Antibodies Using Affinity Chromatography
The synthetic peptide was synthesized using F-MOC chemistry. Half of it had
cysteine added to the N-terminal end. The amino terminus was coupled
through the sulfhydryl group to an affinity column. Once this affinity
column was made, 80 mls of rabbit serum containing polyclonal antibody to
OPF was ammonium sulphate precipitated, dialyzed, and subjected to the
peptide affinity column. It was eluted with 0.2M glycine (pH 2.8) and
approximately 3 mg of purified antibody was retrieved.
EXAMPLE 7
Preparation of Polyclonal antibodies to OPF
Rabbits (male, 6 to 8 week-old) were subcutaneously injected (10
sites/animal, 100 .mu.l/site) with 1.3 mg keyhole lympet hemocyanin
(KLH)-conjugated synthetic 17-mer peptide and 2 ml complete Freund's
adjuvant. Four weeks after the first immunization, animals were boosted
intramuscularly with 1.3 mg KLH-conjugated synthetic 17-mer peptide and 2
ml incomplete Freund's adjuvant 3 times. At each booster, blood was drawn
from an ear vein and tested for its titer against the synthetic 17-mer
peptide by ELISA.
EXAMPLE 8
Affinity Purification of OPF
An alternative method for purification of OPF is to utilize polyclonal or
monoclonal antibodies for affinity purification. Polyclonal or monoclonal
antibodies were used in an affinity column to absorb biological activity
from MH-85 CM, which was eluted from the affinity column with 4 mls of 1M
acetic acid. The eluted fractions are highly enriched in OPF and can be
chromatographed on a reverse-phase high performance liquid chromatography
column to assess the purity.
The purified antibody prepared as in Example 7 was coupled through the
amine group to a second affinity matrix. The binding capacity of the
17-mer affinity column was greater than 3 .mu.g. The antibody was eluted
from the column with 0.2M glycine at low pH (pH 2.8). Next, 500 ml of
MH-85 CM was processed through this antibody affinity column and no
activity was present in the flow-through fraction. The bound fraction was
eluted with two types of elution buffer, 0.1-0.2M glycine and 1M acetic
acid. ELISA assay of the eluates demonstrated that 1M acetic acid was more
effective at eluting the immunoreactive material than the 0.2M glycine.
Therefore, acetic acid is a preferred eluting buffer.
EXAMPLE 9
Preparation of Monoclonal Antibodies to OPF
Monoclonal antibodies (MAb) to OPF were made using an in vitro immunization
technique (Van Ness et al, (1983) Nature 301: 425-427. Spleen cells of 8
to 12 week-old female Balb/C mice were immunized with 250 pg synthetic
17-mer peptide in the presence of 100 .mu.g
N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP, Sigma), 125 .mu.g
lipopolysaccharide (LPS, Difco) and 500 .mu.l culture supernatants of
concanavalin A (50 .mu.g/ml)-stimulated spleen cells (5.times.10.sup.6
/ml) in 5 ml IMDM supplemented with 20% FBS in 6-well plates for 4 days.
The immunized spleen cells were then hybridized with mouse myeloma FO
cells (kindly provided by Dr. Eguchi, Kaneka, Japan) at a ratio of 2:1 in
the presence of 50% polyethylene glycol (1500, Boehringer-Manheim). After
the hybridization, the cells were suspended in 120 ml IMDM supplemented
with 10% FBS and 0.5 ml cell suspension were inoculated onto each well in
48-well plates. After 24 hours, 5.times.10.sup.5 /well thymocytes in 0.5
ml HAT medium were plated onto each well as a feeder layer. The plates
were cultured for 14 days in HAT medium (Flow) and then for 7 to 10 days
in HT medium (Flow). The cells were fed fresh medium every 2 days. The
culture supernatants harvested from the wells in which growing hybridoma
cells cover 50% of the surface area were screened for their cross
reactivity with the synthetic 17-mer peptide by enzyme-linked
immunosorbent assay (ELISA). FIG. 8 demonstrates that at least 3 of the
monoclonal antibodies neutralized (inhibited) the biological activity of
OPF. Medium from hybridoma cells producing monoclonal antibodies
designated 2B7, 3E3, 3D5 and 3E6 was diluted 1:10 in TRAP incubation
buffer and incubated with an equal volume of 10% MH-85 CM. As demonstrated
on FIG. 8, antibodies designated 2B7, 3E3, 3D5 neutralized the OPF
activity in MH-85 CM. Since these antibodies were derived to the synthetic
peptide, this data further comfirms that the amino acid sequence in the
synthetic peptide confers the biological activity present in MH-85 CM.
EXAMPLE 10
Enzyme-linked immunosorbent assay (ELISA) for OPF
The ELISA plates (Nunc) were coated with 100 ng/50 .mu.l/well synthetic
17-mer peptide overnight at 4.degree. C., washed with 200 .mu.l/well
phosphate-buffered saline (PBS) twice, incubated with 200 .mu.l/well
blocking buffer (1% bovine serum albumin in PBS) for 1 hour at 37.degree.
C. and rinsed with 250 .mu.l washing buffer (0.05% Tween 20 in PBS). The
plates were then incubated with 200 .mu.l/well hybridoma culture
supernatants to be tested for overnight at 4.degree. C. and rinsed with
250 .mu.l/well washing buffer 3 times. After rinsing, the plates were
incubated with 200 .mu.l/well peroxidase-conjugated goat anti-mouse
immunoglobulin G antibodies for 1 hour at 37.degree. C., rinsed with 250
.mu.l/well washing buffer 5 times and incubated with 200 .mu.l/well
0-phenylenediamine dihydrochloride (OPD) solution (4 mg OPD and 10 .mu.l
30% hydrogen peroxide in 10 ml water) for 10 to 20 minutes. The reaction
was stopped by adding 80 .mu.l/well 2.5M sulfuric acid. The absorbance was
read at 492 nm. The limits of detection in this assay are 50 pg/ml.
DEPOSIT OF STRAINS USEFUL IN PRACTICING THE INVENTION
Deposits of biologically pure cultures of the following strains were made
under the Budapest Treaty with the American Type Culture Collection, 12301
Parklawn Drive, Rockville, Md., The accession numbers indicated were
assigned after successful viability testing, and the requisite fees were
paid.
Access to said cultures will be available during pendency of the patent
application to one determined by the Commissioner of the United States
Patent and Trademark Office to be entitled thereto under 37 C.F.R.
.sctn.1.14 and 35 U.S.C. .sctn.122, or if and when such access is required
by the Budapest Treaty. All restriction on availability of said cultures
to the public will be irrevocably removed upon the granting of a patent
based upon the application and said cultures will remain permanently
available for a term of at least five years after the most recent request
for the furnishing of samples and in any case for a period of at least 30
years after the date of the deposits. Should the cultures become nonviable
or be inadvertantly destroyed, they will be replaced with viable
cultures(s) of the same taxonomic description.
______________________________________
Strain/Plasmid ATCC No. Deposit Date
______________________________________
MH-85 CRL 10833 July 23, 1991
OPF3E3 HB10534 July 23, 1991
______________________________________
One skilled in the art will readily appreciate the present invention is
well adapted to carry out the objects and obtain the ends and advantages
mentioned, as well as those inherent therein. The peptides, antibodies,
methods, procedures and techniques described herein are presented as
representative of the preferred embodiments, or intended to be exemplary
and not intended as limitations on the scope of the present invention.
Changes therein and other uses will occur to those of skill in the art
which are encompassed within the spirit of the invention or defined by the
scope of the appended claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 3
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AlaValGlnArgTyrLeuValLeuGlnGlyValSerProAlaGlnLeu
151015
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
LysAlaValGlnArgTyrLeuValLeuGlnGlyValSerProAlaGln
151015
Leu
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AARGCNGTNCARMGNTAYYTNGTNYTNCARGGNGTNWSNCCNGCNCARYTN51
__________________________________________________________________________
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